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results from further SAR studies to investigate structural features
References and notes
in the pendant aromatic group that confer slow offset through high
potency in the purine series are shown in Table 4.
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Presence of an R4 substituent in combination with a methyl at
the R3 position (23–26) increased the chances of compounds
appearing insurmountable in the Schild analysis. All compounds
profiled from the pyrazolopyrimidine series, where R3 substitution
is not possible were found to have surmountable profiles (<50%
suppression of maximal response—data not shown). Only one
example screened with both R3 and R4 substituents present (10)
had <50% suppression of maximal response, suggesting that there
were further subtleties to the SAR. It was apparent that increased
suppression of the maximal response was not driven purely by in-
creased lipophilicity, as evidenced by compounds 10 and 25 having
similar c log P, but quite different abilities to suppress the maximal
response.
Compounds with suppression of >75% of the maximal response
(23 and 25) were progressed to full kinetic profiling using the
Motulsky and Mahan methodology. This revealed two compounds
bearing substituents at both R3 and R4 positions did that indeed
have sub-nanomolar potency (Table 5).
Compounds 9, 22, 23 and 25 were progressed into rat pharmaco-
kinetic studies (Table 6). All compounds had low to moderate clear-
ance leading to half lives in rat from 2.7 to 11 h.
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9. CRF-1 functional activity was measured using CHO cells (Cell Sciences
SNB0000377) expressing recombinant human CRF-1 receptor grown in
DMEM:F12 (1:1) media containing 10% (V/V) Foetal Bovine Serum (PAA),
400 lg/ml Geneticin (GIBCO-BRL 10131-027) and 1% (V/V) Glutamax in a cell
incubator at 37 °C, 5% CO2 to 70% confluence. 10 nM FAC hCRF was added with
test compound to 10,000 cells/well in Phosphate Buffered Saline containing
500 lM FAC IBMX. The functional response was measured using DiscoveRx
HitHunter cAMP II Assay kit (Amersham Biosciences—90-0034-03). Each
compound was tested multiple times using a 0.5 log serial dilution dose
response with a top final assay concentration of 20 lM. The % response of the
Compound 25was also dosed orally as a suspension to rats (8 mg/
kg) and showed good oral bioavailability (F = 52%). Receptor occu-
pancy studies on compounds 22 and 25 are reported elsewhere,13
and demonstrate that these compounds achieve high receptor occu-
pancy at the CRF-1 receptor in the CNS.
Potent purine CRF-1 antagonists have been identified. Pharmaco-
logical profiling has shown that our primary functional assay proto-
col was unable to identify sub-nanomolar compounds due to the
slow offset properties conferred by high potency compounds.
Surmountability in a Schild analysis was used as a secondary screen
to identify the most potent compounds, with promising compounds
followed up using the methodology of Motulsky and Mahan to
determine kinetic parameters and calculate a kinetically derived
Kd. The compounds in Table 6 provide a useful set of potent CRF-1
antagonists with good in vivo pharmacokinetic profiles and a rage
of dissociative half-lives to allow exploration of the effects of bind-
ing kinetics on efficacy and safety of purine CRF-1 antagonists.
test compound at different test doses were then fitted to a 4-parameter logistic
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IC50 using the Cheng and Prussoff relationship and the EC50 for the agonist does
response curve which was determined on the same day.
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figures of <30 are generally regarded as having a good chance of translating to
moderate to low in vivo clearance, and Clint figures of >70 are likely to translate
to rapid in vivo clearance.
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Acknowledgements
We acknowledge Denise Harding, Laia Malet and Adam Stennett
for compound synthesis, Simeon Ramsay, Margaret Jackson, Nick
Edmunds, Jo Mulgrew, Graham Baker, David Winpenny, Pauline
Carnell and Rachel Russell for biological data and Joanne Phipps
and Satish Dayal for ADME studies.
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Vauquelin, G.; Van Liefde, I.; Birzbier, B. B.; Vanderheyden, P. M. L. Fundam. Clin.
Pharmacol. 2002, 16, 263.